DDX41 haploinsufficiency causes inefficient hematopoiesis under stress and cooperates with p53 mutations to cause hematologic malignancy

Germline heterozygous mutations in DDX41 predispose individuals to hematologic malignancies in adulthood. Most of these DDX41 mutations result in a truncated protein, leading to loss of protein function. To investigate the impact of these mutations on hematopoiesis, we generated mice with hematopoietic-specific knockout of one Ddx41 allele. Under normal steady-state conditions, there was minimal effect on lifelong hematopoiesis, resulting in a mild yet persistent reduction in red blood cell counts. However, stress induced by transplantation of the Ddx41+/− BM resulted in hematopoietic stem/progenitor cell (HSPC) defects and onset of hematopoietic failure upon aging. Transcriptomic analysis of HSPC subsets from the transplanted BM revealed activation of cellular stress responses, including upregulation of p53 target genes in erythroid progenitors. To understand how the loss of p53 affects the phenotype of Ddx41+/− HSPCs, we generated mice with combined Ddx41 and Trp53 heterozygous deletions. The reduction in p53 expression rescued the fitness defects in HSPC caused by Ddx41 heterozygosity. However, the combined Ddx41 and Trp53 mutant mice were prone to developing hematologic malignancies that resemble human myelodysplastic syndrome and acute myeloid leukemia. In conclusion, DDX41 heterozygosity causes dysregulation of the response to hematopoietic stress, which increases the risk of transformation with a p53 mutation.

For in vitro measurement of protein synthesis, Click-iT HPG Alexa-Fluor 594 Protein Synthesis Assay was used (Thermo).
Cells were transduced with viral supernatant containing 0.8µg/ml polybrene for 24hrs.Expression of GFP was determined using flow cytometry.

Single-cell RNA Sequencing
The scRNA-Seq assay was performed according to the manufacturer's instructions (Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for Cell Surface Protein, 10x Genomics).
Briefly, Total-Seq B antibody-labeled cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate a gel bead-in-emulsion (GEM).The poly-A RNA from the cell lysate contained in every GEM was reverse transcribed into cDNA, adding an Illumina TruSeq R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode.The DNA conjugated to the Total-Seq B antibodies (Feature Barcodes aka FBs) was also barcoded by adding an Illumina Nextera R1, UMI, and the 10x Barcode.The cell barcoded molecules were then cleaned up with Silane DynaBeads and amplified using 14 PCR cycles.Size selection using SPRIselect reagent was performed post amplification to separate full-length cDNA from FBs. Next, full-length, barcoded cDNA was then enzymatically fragmented, sized-selected, adapterligated, and amplified for library construction.During the library construction, P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 were added.Separately, FBs were prepared into library constructs by incorporating P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 via PCR.Samples were pooled and run on the NovaSeq 6000 sequencer with a S4 flow cell using the following sequencing parameters: R1: 28 cycles, i7: 10 cycles, i5: 10 cycles, R2: 90 cycles.Sequencing data processing and sample demultiplexing was done using cellranger multi 30 .
Matrices were processed with Seurat using standard normalization procedures 31 .Only cells with less than or equal to 5% mitochondrial gene content and number of genes between 500 and 7,500 were kept.Clustering of cells based on graphical features was done using the provided Louvain algorithm.We used multiple resolutions to find the cluster structure that best captures the expected group of hematopoietic cells.Clusters were named based on the expression of lineage-specific genes (markers) using a single-cell atlas of mouse hematopoiesis 32 .
Differentially expressed genes between clusters were found with the DESeq2 method 33 .An average expression of each gene from all cells in each cluster was calculated for this purpose.

Whole Exome Sequencing on mouse bone marrow
DNA was isolated from BM mononuclear cells using the Zymo Quick-DNA kit.Mouse Exome was enriched using Twist Mouse Exome probe set following manufacturer's protocols (Twist Biosciences).Samples were sequenced on an Illuumina NovaSeq.FASTQ files were aligned to mouse mm10/GRCm38 genome.Variants were called with DeepVariant and variant annotations with snpEff 34,35 .